Effective Treatment For Multiple Myeloma Research Articles

Disruption of DNA-PKcs-mediated cGAS retention on damaged chromatin potentiates DNA damage-inducing agent-induced anti-multiple myeloma activity.

Targeting DNA damage repair factors, such as DNA-dependent protein kinase catalytic subunit (DNA-PKcs), may offer an opportunity for effective treatment of multiple myeloma (MM). In combination with DNA damage-inducing agents, this strategy has been shown to improve chemotherapies partially via activation of cGAS-STING pathway by an elevated level of cytosolic DNA. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated into the cytoplasm upon DNA damage, leading to cGAS-STING activation. We examined the role of DNA-PKcs inhibition on cGAS-STING-mediated MM chemosensitivity by performing mass spectrometry and mechanism study. Here, we found DNA-PKcs inhibition potentiated DNA damage-inducing agent doxorubicin-induced anti-MM effect by activating cGAS-STING signaling. The cGAS-STING activation in MM cells caused cell death partly via IRF3-NOXA-BAK axis and induced M1 polarization of macrophages. Moreover, this activation was not caused by defective classical non-homologous end joining (c-NHEJ). Instead, upon DNA damage induced by doxorubicin, inhibition of DNA-PKcs promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of cytosolic cGAS and DNA, activating cGAS-STING. Inhibition of DNA-PKcs could improve the efficacy of doxorubicin in treatment of MM by de-sequestrating cGAS in damaged chromatin.

Disruption of DNA-PK-Mediated Cgas Retention on Damaged Chromatin Potentiates Doxorubicin-Induced Cgas/Sting-Dependent Anti-Multiple Myeloma Activity

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM with multidrug resistance or extensive extramedullary plasmacytoma are difficult to manage and often treated with DNA damage-inducing chemotherapies, including topoisomerase II inhibitor doxorubicin. However, the efficacy of this treatment is still limited and requires further improvement. Targeting DNA damage response (DDR) may offer an opportunity for effective treatment of MM. In particular, in combination with DNA damage-inducing agents, this strategy has shown to improve chemo-radiotherapies in part by cGAS-STING-mediated innate immune response, which could be directly activated by an elevated level of cytosolic DNA due to DNA repair inhibition. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated to the cytoplasm upon DNA damage, leading to cGAS-STING activation. Here, after testing the potential of several DDR inhibitors in sensitizing MM cells (MMCs) to doxorubicin, we found that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of classical non-homologous end joining (c-NHEJ) repair, using DNA-PKcs inhibitor NU7441, synergistically enhanced doxorubicin-induced anti-MM effect by activation of cGAS-STING pathway, the combination index values at different combination doses were all less than 0.85. Knocking out either cGAS or STING in ARP-1 cells significantly reduced not only the transcription of IFNB1 and multiple ISGs but also the proportion of NU7441/doxorubicin-induced apoptotic cells. Stably overexpressing STING in U266 and RPMI-8226 cells significant upregulated the IFNB1 and ISGs levels, and made MMCs more susceptible to NU7441/doxorubicin-induced inhibition of cell proliferation. Despite a strong type I interferon (IFN) response induced by activation of cGAS-STING, apoptosis of MMCs caused by activated cGAS-STING was mediated in part by IRF3-NOXA-BCL2 axis, which is independent of type I IFN response. In addition to inducing intrinsic apoptosis of MMCs, activation of cGAS-STING signaling by NU7441/doxorubicin in MMCs also induced M1 polarization of macrophages (Mφs), which increased M1 marker CD86 of Mφs but decreased M2 marker CD163 and CD206, and reduced the IL-10 concentration secreted by Mφs, thus suppressing tumor-protecting potential of M2-like Mφs and improving bortezomib-induced apoptosis of MMCs. Mechanistic studies showed that the activation of cGAS-STING signaling was not due to the defective c-NHEJ following continuous DNA-PKcs inhibition, because NU7441 did not enhance the damaged DNA accumulation after doxorubicin induction detected by γH2AX immunoblot analysis and alkaline comet assay. Besides, deletion of XRCC4 in ARP-1 cells to directly impair c-NHEJ did not enhance doxorubicin-induced activation of cGAS-STING signaling, further excluding a role for impaired c-NHEJ. Instead, we found that cGAS was mainly localized in the nucleus and chromatin-bound in MMCs under mock treatment, and doxorubicin induced DNA damage and mobilized inactive cGAS into cytoplasm. Both mass spectrometry data and immunoblotting of cGAS immunocoprecipitates showed that cytoplasmic cGAS was associated with DNA-PK complex and nucleosomal histone H2A-H2B heterodimer, and DNA-PKcs inhibition promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of free cytosolic cGAS and DNA, activating cGAS-STING. Disruption of cytoplasmic cGAS-nucleosome binding by mutating cGAS R236 or R255 sites to E abolished the effect of NU7441 on doxorubicin-induced activation of cGAS-STING signaling. Analysis of Datasets MMRF CoMMpass and GSE2658 showed that MM patients with higher DNA-PKcs expression showed poorer prognosis than those with lower expression. Additionally, the DNA-PKcs expression was elevated with MM disease progression according to Datasets GSE5900, GSE13591 and MMRF CoMMpass, these indicated a possible involvement of DNA-PKcs in MM progression and provided a basis for targeting DNA-PKcs in MM treatment. Taken together, our study suggests that DNA-PKcs may help maintain the cGAS sequestration in damaged chromatin. Inhibition of DNA-PKcs may consequently disrupt this sequestration, inducing activation of cGAS-STING signaling and improving the efficacy of doxorubicin in treating MM.

Anti-BCMA surface engineered biomimetic photothermal nanomissile enhances multiple myeloma cell apoptosis and overcomes the disturbance of NF-κB signaling in vivo

Conventional chemotherapy for multiple myeloma (MM) faces the challenges of a low complete remission rate and transformation to recurrence/refractory. The current MM first-line clinical drug Bortezomib (BTZ) faces the problem of enhanced tolerance and nonnegligible side effects. B cell maturation antigen (BCMA), for its important engagement in tumor signaling pathways and novel therapy technologies such as Chimeric antigen receptor T-Cell immunotherapy (CAR-T) and Antibody Drug Conjugate (ADC), has been identified as an ideal target and attracted attention in anti-MM therapy. Emerging nanotechnology provided feasible methods for drug delivery and new therapeutic strategies such as photothermal therapy (PTT). Herein, we developed a BCMA-Targeting biomimetic photothermal nanomissile BTZ@BPQDs@EM @anti-BCMA (BBE@anti-BCMA) by integration of BTZ, black phosphorus quantum dots (BPQDs), Erythrocyte membrane (EM) and BCMA antibody (anti-BCMA). We hypothesized that this engineered nanomissile could attack tumor cells in triple ways and achieve effective treatment of MM. Consequently, the intrinsic biomimetic nature of EM and the active targeting property of anti-BCMA enhanced the accumulation of therapeutic agents in the tumor site. Besides, owing to the decrease in BCMA abundance, the potential apoptosis-inducing ability was revealed. With the support of BPQDs' photothermal effect, Cleaved-Caspase-3 and Bax signal increased significantly, and the expression of Bcl-2 was inhibited. Furthermore, the synergistic photothermal/chemo therapy can effectively inhibit tumor growth and reverse the disorder of NF-κB in vivo. Importantly, this biomimetic nanodrug delivery system and antibody induced synergistic therapeutic strategy efficiently killed MM cells with ignorable systemic toxicity, which is a promising method for the future anticancer treatment of hematological malignancies in clinics.

SUMOylation inhibition overcomes proteasome inhibitor resistance in multiple myeloma.

Proteasome inhibition is a highly effective treatment for multiple myeloma (MM). However, virtually all patients develop proteasome inhibitor resistance, which is associated with a poor prognosis. Hyperactive small ubiquitin-like modifier (SUMO) signaling is involved in both cancer pathogenesis and cancer progression. A state of increased SUMOylation has been associated with aggressive cancer biology. We found that relapsed/refractory MM is characterized by a SUMO-high state, and high expression of the SUMO E1-activating enzyme (SAE1/UBA2) is associated with poor overall survival. Consistently, continuous treatment of MM cell lines with carfilzomib (CFZ) enhanced SUMO pathway activity. Treatment of MM celllines with the SUMO E1-activating enzyme inhibitor subasumstat (TAK-981) showed synergy with CFZ in both CFZ-sensitive and CFZ-resistant MM cell lines, irrespective of the TP53 state. Combination therapy was effective in primary MM cells and in 2 murine MM xenograft models. Mechanistically, combination treatment with subasumstat and CFZ enhanced genotoxic and proteotoxic stress, and induced apoptosis was associated with activity of the prolyl isomerase PIN1. In summary, our findings reveal activated SUMOylation as a therapeutic target in MM and point to combined SUMO/proteasome inhibition as a novel and potent strategy for the treatment of proteasome inhibitor-resistant MM.

Quadruple gene-engineered natural killer cells enable multi-antigen targeting for durable antitumor activity against multiple myeloma

Allogeneic natural killer (NK) cell adoptive transfer is a promising treatment for several cancers but is less effective for the treatment of multiple myeloma. In this study, we report on quadruple gene-engineered induced pluripotent stem cell (iPSC)-derived NK cells designed for mass production from a renewable source and for dual targeting against multiple myeloma through the introduction of an NK cell-optimized chimeric antigen receptor (CAR) specific for B cell maturation antigen (BCMA) and a high affinity, non-cleavable CD16 to augment antibody-dependent cellular cytotoxicity when combined with therapeutic anti-CD38 antibodies. Additionally, these cells express a membrane-bound interleukin-15 fusion molecule to enhance function and persistence along with knock out of CD38 to prevent antibody-mediated fratricide and enhance NK cell metabolic fitness. In various preclinical models, including xenogeneic adoptive transfer models, quadruple gene-engineered NK cells consistently demonstrate durable antitumor activity independent of exogenous cytokine support. Results presented here support clinical translation of this off-the-shelf strategy for effective treatment of multiple myeloma.

Early and late relapses of multiple myeloma after autologous haematopoietic stem cell transplantation

Introduction. Autologous haematopoietic stem cell transplantation (auto-HSCT) is a highly effective treatment for multiple myeloma (MM). Auto-HSCT allows a signifi cant improvement of haematological response leading to higher overall survival and quality of life in MM patients. Nonetheless, the majority of patients develop relapse.Aim — a comparison of clinical MM relapses developing at variant terms after auto-HSCT.Patients and methods. A retrospective study enrolled 65 MM patients aged between 39 and 64 years. All patients had auto-HSCT during 2009–2019, all had achieved complete response (CR) or very good partial response (VGPR) and all since developed immunochemical MM relapse in laboratory evidence. Patients were divided in two cohorts by relapse term, the early (within 12 months of auto-HSCT) and late relapse.Results. Early immunochemical relapse was diagnosed in 13 (20 %), late relapse — in 52 (80 %) patients. The dependence between relapse term and depth of post-auto-HSCT antitumour response has been determined. The proportion of CR patients was signifi cantly higher in late than in early relapse (55.8 vs. 23 %). In follow-up, 60 patients (92.3 %) were initiated on antirelapse therapy, all early relapse and 90.3 % late relapse patients. On day +100 of auto-HSCT, CR patients had later relapse vs. VGPR individuals (median 24 vs. 19.9 months, p = 0.08) with signifi cantly weaker paraprotein secretion resembling the clinical course of monoclonal gammopathy of unclear signifi cance (MGUS).Conclusion. Auto-HSCT allows long-term control of the disease. A signifi cant prognostic factor is antitumour response on +100 day of auto-HSCT. Patients attaining CR have later relapse progressing in a MGUS-like manner. Patients with late indolent relapse can be managed long-term without antitumour therapy.

Is It Time for a More Holistic Approach to the Treatment of Multiple Myeloma?

In the UK, approximately 5820 new cases of multiple myeloma (MM) are diagnosed each year. This number has increased by a third since the early 1990s. Considerable progress has been achieved in our ability to treat MM as a result of the development of new chemotherapeutic agents. MM is a disease more commonly seen in elderly individuals who frequently have pre-existing co-morbidities and are subject to social pressures that impact adversely on their quality of life (QOL). As their lives are extended by more effective treatment of MM, there is a greater need to address such issues. This review will focus on the holistic needs of a patient with MM, and how all members of the multidisciplinary team have a role. The aim is to advocate for centres to support MM patients to live well with their condition.

Efficacy of Modified Vrd-Lite for Transplant Ineligible Multiple Myeloma

BACKGROUND: High dose chemotherapy followed by autologous peripheral blood stem cell transplantation is an effective treatment for multiple myeloma. However, many patients with newly diagnosed multiple myeloma are transplant-ineligible because of their age and complications, result in a poorer prognosis than transplant-eligible patients. Furthermore, many of them cannot complete normal chemotherapy because of low tolerability. Here, we investigated the efficacy and safety of modified bortezomib with lenalidomide and dexamethasone (mVRD-lite) for transplant-ineligible patients with newly diagnosed multiple myeloma. STUDY DESIGN: A retrospective observational analysis was performed on patients who received mVRd-lite for the first line chemotherapy between Jan. 2016 and Mar. 2020 in our hospital. Patients who received high dose dexamethasone to reduce tumor burden, and patients who received bortezomib with dexamethasone or lenalidomide with dexamethasone as a reduction regimen of mVRd-lite were also included. We evaluated ORR, OS, PFS and adverse effect. mVRD-lite at first was administered over a 28-day cycle. Bortezomib 1.3 mg/m2 weekly was administered subcutaneously on days 1, 8, 15 and 22. Lenalidomide 15 mg was given orally 18 days, omitted on days 1, 8, 15, which are the days of bortezomib administration. Dexamethasone 20 mg was given orally on days 1, 2, 8, 9, 15, 16, 22, which are the day of and day after bortezomib. We also reviewed patients background, especially complication of light-chain amyloidosis and considered the impact of cardiac amyloidosis on patient prognosis. This study was conducted with the permission of the Ethics Review Board in our hospital. RESULTS: The subjects analyzed totaled 40 transplant-ineligible patients. 11(27.5%) patients were AL amyloidosis associated with multiple myeloma and 8(20%) patients had cardiac amyloidosis. Median age at diagnosis was 73 (range 48-86) and Male:Female=1:1. Most of them were judged inadequate to transplantation due to their age, general condition, or complication. One patient was ruled unfit to transplantation, because of his refusion. The Revised International Staging System (R-ISS) were I in 5 (12.5%), II in 25 (62.5%) and III in 8 (20%). 5(25%) patients switched to maintenance therapy. 17(42.5%) patients discontinued treatment, because of adverse effect (cardiac failure 4 ; two of them combined with cardiac amyloidosis, rash 4, peripheral neuropathy 3, infection 3, etc). 2(5%) patients died during treatment by mVRd-lite, because of Grade 4 adverse effect, such as pneumonia. 11(27.5%) patients died during observation period and causes of death were primary disease and TRM. 1(2.5%) patient was died of heart failure associated with cardiac amyloidosis. The overall response rate(ORR) during treatment period of mVRd-lite was obtained in 34(85%), including sCR in 5 (12.5%), VGPR in 13 (32.5%) and PR in 14(30%). 2(5%) patients achieved MRD negative. SD were observed in 3(7.5%) patients. 3(7.5%) patients were not evaluated efficacy because of treatment interruption by adverse effect. Overall survival rate at two year is 64.3%, median OS was not reached, at a median follow-up of 20 months. CONCLUSIONS: Transplant-ineligible multiple myeloma patients are associated with poor prognosis. Modified RVD-lite is one of the appropriate therapeutic options, in the transplant-ineligible multiple myeloma patients. Twenty-five percent of patients with cardiac amyloidosis had treatment discontinued due to cardiac complications. Further study is needed for treatment of patients with multiple myeloma complicated with cardiac amyloidosis. Disclosures Ishida: Janssen: Speakers Bureau; Celgene: Speakers Bureau; Ono pharmaceutical co: Speakers Bureau; Takeda pharmaceutical co: Speakers Bureau. Nashimoto:Janssen: Speakers Bureau; Celgene: Speakers Bureau. Tsukada:Takeda pharmaceutical co: Speakers Bureau. Suzuki:Takeda, Amgen, Janssen and Celgene: Consultancy; Takeda, Celgene, ONO, Amgen, Novartis, Sanofi, Bristol-Myers Squibb, AbbVie and Janssen: Honoraria; Bristol-Myers Squibb, Celgene and Amgen: Research Funding.

An Iterative Formative Evaluation of Medical Education for Multiple Myeloma Patients Receiving Autologous Stem Cell Transplant.

Multiple myeloma (MM) is an incurable cancer characterized by abnormal plasma cells in the bone marrow, resulting in increased risk of infection. Autologous stem cell transplant (ASCT) is the most effective treatment for MM, but successful transplant requires the patient and caregiver to learn and independently implement medical tasks. The Ready for Transplant (R4T) ethnographic-based formative evaluation includes evaluation of ongoing patient education and the addition of novel supplemental educational videos that patients can review before in-person transplant education. To evaluate R4T, a formative evaluation informed by utilization-orientated evaluation theories, including over 150hours of ethnographic observation of nurse/patient education (N= 70), plus retrospective and prospective patient (N= 35) and clinician interviews (N= 7) was conducted over 18months. Through thematic analysis, barriers and facilitators of the patient's ability to process education surrounding ASCT were identified. Barriers included anxiety surrounding what to expect in ASCT, anxiety surrounding what to expect at the education visit, overwhelming volume of information, lack of medical expertise, and disengaged patients, while facilitators included large social networks willing to help and clinician adjusting script to meet unique needs of the patient. This manuscript represents an iterative method for improving on education that people with MM receive surrounding ASCT. Ultimately, the supplemental video-based education was created to address modifiable social and psychological factors by providing generalized information that could then be tailored during in-person meetings to meet each patients' individual needs.

Multiple myeloma cells are exceptionally sensitive to heat shock, which overwhelms their proteostasis network and induces apoptosis

Proteasome inhibitors, such as bortezomib (BTZ), are highly effective and widely used treatments for multiple myeloma. One proposed reason for myeloma cells' exceptional sensitivity to proteasome inhibition is that they produce and continually degrade unusually large amounts of abnormal immunoglobulins. We, therefore, hypothesized that, heat shock may also be especially toxic to myeloma cells by causing protein unfolding, increasing further the substrate load on proteasomes, and, thus, putting further stress on their capacity for protein homeostasis. After a shift from 37 to 43 °C, all four myeloma lines studied underwent extensive apoptosis in 4 h, unlike 13 nonmyeloma cell lines, even though the myeloma cells induced heat-shock proteins and increased protein degradation similar to other cells. Furthermore, two myeloma lines resistant to proteasome inhibitors were also more resistant to 43 °C. Shifting myeloma cells to 43, 41, or 39 °C (which was not cytotoxic) dramatically increased their killing by proteasome inhibitors and inhibitors of ubiquitination or p97/VCP. Combining increased temperature with BTZ increased the accumulation of misfolded proteins and substrate load on the 26S proteasome. The apoptosis seen at 43 °C and at 39 °C with BTZ was mediated by caspase-9 and was linked to an accumulation of the proapoptotic Bcl-2-family member Noxa. Thus, myeloma cells are exceptionally sensitive to increased temperatures, which greatly increase substrate load on the ubiquitin-proteasome system and eventually activate the intrinsic apoptotic pathway. Consequently, for myeloma, mild hyperthermia may be a beneficial approach to enhance the therapeutic efficacy of proteasome inhibitors.

Genome-wide screening reveals a role for subcellular localization of CRBN in the anti-myeloma activity of pomalidomide

Pomalidomide, a derivative of thalidomide, is an effective treatment for multiple myeloma. The drug exerts its effects through CRBN, a component of the E3 ubiquitin ligase complex CRL4CRBN. To search for novel factors involved in the anti-cancer activity of pomalidomide, we performed a genome-wide shRNA library screen and identified 445 genes as those affecting pomalidomide sensitivity. Genes encoding components of the ubiquitin-proteasome pathway, such as subunits of the CRL4CRBN complex, the COP9 signalosome, and the 26S proteasome, were among the pomalidomide-affecting genes. Karyopherin beta 1 (KPNB1) was identified as a novel pomalidomide-affecting gene. KPNB1 was required for the nuclear import of CRBN and for the CRBN-directed, pomalidomide-dependent degradation of a clinically relevant substrate, the transcription factor Aiolos. By contrast, the cytoplasmic translation factor GSPT1 was degraded following treatment with the thalidomide derivative CC-885 only when CRBN was present in the cytoplasm, indicating that subcellular distribution of CRBN is critical for the efficacy of thalidomide-based medications.

CD44-targeted vesicles encapsulating granzyme B as artificial killer cells for potent inhibition of human multiple myeloma in mice

Multiple myeloma (MM) is a malignant blood cancer homing in bone marrow that is particularly hard to treat. The effective treatment for MM shall be not only MM-selective but also capable of homing to bone marrow. Herein, we report on hyaluronic acid-directed reduction-responsive chimaeric polymersomes encapsulating a key player in the NK cells, granzyme B (HA-RCP-GrB) as an artificial killer cell for targeted protein therapy of MM. Interestingly, HA-RCP-GrB displayed high MM-targetability and anti-MM activity with a remarkably low IC50 of 8.1 nM toward CD44 overexpressing LP1 human MM cells. The in vivo biodistribution studies using Cy5-labeled cytochrome C as a model protein demonstrated significantly enhanced accumulation of HA-RCP in the subcutaneous LP1 tumor as well as in the bone marrow of orthotopic LP1 MM model compared with the non-targeted RCP counterparts, confirming that HA-RCP possesses MM-selectivity and is able to deliver proteins to the bone marrow. In accordance, HA-RCP-GrB exerted significantly better suppression of subcutaneous LP1 tumor than the non-targeted RCP-GrB. More interestingly, in the orthotopic LP1 MM-bearing mice, HA-RCP-GrB led to significant survival benefits and less body weight loss over PBS and RCP-GrB. μCT analyses, H&E and TRAP staining revealed that mice treated with HA-RCP-GrB had greatly reduced osteolysis and proliferation of atypical plasma cells in the bone marrow. HA-RCP-GrB has emerged as a novel and effective treatment for multiple myeloma.

A review on the potential of Bruton’s tyrosine kinase (Btk) inhibitor – Ibrutinib for treatment of Multiple Myeloma (MM)

Multiple myeloma (MM) is characterized by the over-production of monoclonal plasma cells that eventually become malignant in the bone marrow. MM remains as an incurable cancer, but it can be treated through chemotherapy. Nonetheless, research on novel therapies for effective treatment of MM is ongoing and in this case the involvement of Bruton’s tyrosine kinase (Btk) in B-cell malignancies has made it one of the new therapeutic targets. In MM patients, it has been reported that the expression of Btk was elevated and this could potentially contribute to chemoresistance indirectly via enhancement of cell proliferation and survival. Ibrutinib is a highly selective irreversible Btk inhibitor commonly used as treatment for B-cell malignancies such as Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL). With reference to the potential involvement of Btk in MM and current treatment of MCL and CLL using ibrutinib, researchers have begun to examine the effect of ibrutinib treatment on MM. This review provides information on the association of MM and Btk in conjunction with the treatment using ibrutinib. To date, clinical trials of ibrutinib as therapeutic alternative for MM have produced promising results, particularly as combination therapy with other agents such as dexamethasone and carfilzomib. However, there is limited evidence on the Btk mechanisms involved in MM, hence, it is important to further investigate the Btk oncogenic signalling pathway(s) in MM cells in order to establish successful and improved treatment of MM.

Clinical significance of CXCR5-CXCL13 signaling in multiple myeloma.

e19517 Background: Multiple myeloma (MM) is characterized by the neoplastic proliferation of plasma cells resulting in monoclonal immunoglobulins in the blood, bone marrow, and urine (1). According to a 2019 American Cancer Society (ACS) report, MM is now classified as subtypes of non-Hodgkin lymphoma (NHL). ACS expects there will be ~32,110 new MM cases (18,130 in men and 13,980 in women) and ~12,960 (6,990 in men and 5,970 in women) anticipated deaths in 2019. MM is a heterogenous disease; characterizing its molecular phenotypes is important for effective treatment of MM and predicting relapse. CXCL13-CXCR5 interactions are involved in malignancy cell homing, adhesion, signal transduction, and calcium flux, all of which promote MM progression. Methods: We analyzed RNA-sequence data from matched normal, primary and relapsed MM cases. Patient control mRNA samples matched primary tumor and relapse samples were acquired from dbGap (database of Genotypes and Phenotypes) to evaluate the mRNA expression patterns of CXCL13, CXCR5 and associated genes in MM. A bioinformatics strategy was used to integrate published genomic data from MM patients (n = 480) and identify genes associated with CXCL13-CXCR5 signaling. DESeq analysis was used to determine differentially expressed genes between normal tissues, primary tumor and relapse groups. Weighted gene co-expression network analysis (WGCNA) identified clusters of genes significantly associated with the molecular phenotypes of MM. Results: Notably, gene expression driven by NFAT and JUN, known to be activated by the CXCL13-CXCR5 axis, and plasma cell signaling pathways significantly correlated with select MM molecular phenotypes and patient survival. Ingenuity pathway analysis (IPA) was performed to analyze upstream regulators, gene interaction networks and canonical pathways. Taken together, our data show CXCR5-CXCL13 signaling networks are significantly expressed and associated with MM pathogenesis and plasma clonality. Conclusions: This study provides a better understanding of the heterogeneous nature of MM and the role of the CXCL13-CXCR5 axis in MM.

Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma.

Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.

Antioxidant Defenses Confer Resistance to High Dose Melphalan in Multiple Myeloma Cells

Background: Multiple myeloma (MM) is the second most common hematological cancer after lymphoma. It is characterized by the accumulation of clonal malignant plasma cells within the bone marrow. The development of drug resistance remains a major problem for effective treatment of MM. Understand the mechanisms underlying drug resistance in MM is a focal point to improve MM treatment. Methods: In the current study, we analyzed further the role of redox imbalance induction in melphalan-induced toxicity both in human myeloma cell lines (HMCLs) and primary myeloma cells from patients. Results: We developed an in-vitro model of short-term resistance to high-dose melphalan and identified that pretreatment with physiological concentration of GSH protects HMCLs from melphalan-induced cell cycle arrest and cytotoxicity. We validated these results using primary MM cells from patients co-cultured with their bone marrow microenvironment. GSH did not affect the ability of melphalan to induce DNA damages in MM cells. Interestingly, melphalan induced reactive oxygen species, a significant decrease in GSH concentration, protein and lipd oxydation together with NRF2 (NF-E2-related factor 2) pathway activation. Conclusions: Our data demonstrate that antioxidant defenses confers resistance to high dose melphalan in MM cells, supporting that redox status in MM cells could be determinant for patients’ response to melphalan.

Novel Combination Therapy Targeting rDNA Transcription and Histone Deacetylation Provides Effective Treatment for Multiple Myeloma, and Synergises in Bortezomib-Resistant MM

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies.Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs).Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance.Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity.The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response.Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment.We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials.Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma.ReferencesDrygin et al., Cancer Research 2011Bywater et al., Cancer Cell 2012Quin et al, Oncotarget, 2016Devlin et al., Cancer Discovery 2016Hein et al., Blood 2017 DisclosuresHarrison:Janssen-Cilag: Other: Scientific advisory board.

Genome-wide screen identifies cullin-RING ligase machinery required for lenalidomide-dependent CRL4CRBN activity

AbstractLenalidomide mediates the ubiquitination and degradation of Ikaros family zinc finger protein 1 (IKZF1), IKZF3, and casein kinase 1α (CK1α) by facilitating their interaction with cereblon (CRBN), the substrate receptor for the CRL4CRBN E3 ubiquitin ligase. Through this mechanism, lenalidomide is a clinically effective treatment of multiple myeloma and myelodysplastic syndrome (MDS) with deletion of chromosome 5q [del(5q) MDS]. To identify the cellular machinery required for lenalidomide-induced CRL4CRBN activity, we performed a positive selection, genome-scale clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) screen in a lenalidomide-sensitive myeloma cell line. CRBN was the top-ranking gene, with all CRBN-targeting guide RNAs (gRNAs) ranking as the 6 highest-scoring gRNAs. A counterscreen using an IKZF3 degron reporter to assay lenalidomide-induced protein degradation highlighted regulators of cullin-RING ligase neddylation and 2 E2 ubiquitin-conjugating enzymes as necessary for efficient lenalidomide-induced protein degradation. We demonstrated that loss of UBE2M or members of the constitutive photomorphogenesis 9 (COP9) signalosome results in altered neddylation of cullin 4A and impairs lenalidomide-dependent CRL4CRBN activity. Additionally, we established that UBE2D3 and UBE2G1 play distinct roles in substrate ubiquitination by CRL4CRBN, with UBE2D3 acting to prime targets via monoubiquitination and UBE2G1 functioning to extend polyubiquitin chains with lysine 48 linkages. The validation of UBE2D3 and UBE2G1 highlights the functional capacity of CRISPR-Cas9 screening to identify E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase complex pairings. More broadly, these findings establish key proteins required for lenalidomide-dependent CRL4CRBN function in myeloma and inform potential mechanisms of drug resistance.

Nanotherapeutics for multiple myeloma.

Multiple myeloma (MM) is an age-related hematological malignancy with an estimated 30,000 new cases and 13,000 deaths per year. A disease of antibody-secreting malignant plasma B-cells that grow primarily in the bone marrow (BM), MM causes debilitating fractures, anemia, renal failure, and hypercalcemia. In addition to the abnormal genetic profile of MM cells, the permissive BM microenvironment (BMM) supports MM pathogenesis. Although advances in treatment options have significantly enhanced survival in MM patients, transient perfusion of small-molecule drugs in the BM does not provide sufficient residence to enhance MM cell-drug interaction, thus allowing some myeloma cells to escape the first line of treatment. As such, there remains a crucial need to develop advanced drug delivery systems that can navigate the complex BMM and effectively reach the myeloma cells. The high vascular density and spongy nature of bone structure suggest that nanoparticles (NPs) can serve as smart drug-delivery systems capable of extravasation and retention in various BM compartments to exert a durable therapeutic effect. In this focus article, we first summarize the pathophysiology of MM, emphasizing how the BM niche presents serious challenges for effective treatment of MM with small-molecule drugs. We then pivot to current efforts to develop NP-based drug carriers and intrinsically therapeutic nanotherapeutics. The article concludes with a brief perspective on the opportunities and challenges in developing and translating nanotherapeutics to improve the treatment outcomes of MM patients. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Expert consensus for the diagnosis and treatment of patients with renal impairment of multiple myeloma

Renal impairment (RI) is a common complication of multiple myeloma (MM), which is presented as chronic kidney disease (CKD) or acute kidney injury (AKI). The typical pathological feature is cast nephropathy. Presently international system staging (ISS) is used in evaluating MM. Although the classic Durie-Salmon staging system could be still used in clinical practice, it may miss out some patients with renal impairment. For evaluations of RI in MM patients with CKD, it's recommended to assess the estimated glomerular filtration rate (eGFR) by creatinine based formula CKD-epidemiology collaboration (EPI) or modification of diet in renal disease(MDRD) and to stage the renal injuries according to 2013 Kidney Disease Improving Global Outcomes (KDIGO) CKD guidelines. For MM patients with AKI, KDIGO AKI guidelines is recommended for evaluation. Renal biopsy is not a routine procedure in all MM patients. It's necessary for patients presenting with glomerular injuries such as albuminuria > 1 g/24 h to eliminate immunoglobulin associated amyloidosis (AL) and monoclonal immunoglobulin deposition disease (MIDD). The effective treatment of MM can reduce serum light chain concentration and improve renal function. The basis of the RI treatment in MM is bortizomib-based regimen, which does not require dosage adjustment in patients with dialysis or renal insufficiency. Thalidomide and lenalidomide are two major immunomodulators in MM treatment. Thalidomide can be used effectively in RI patients without dosage adjustment while lenalidomide should be used cautiously in patients with mild or moderate RI with dosage adjustment and serum toxicity surveillance. High-dose therapy (HDT) and autologous peripheral blood stem cell transplantation (APBSCT) can be therapeutical options for RI patients younger than 65 y, and they should be considered more prudently in patients with severe renal insufficiency (GFR<30 ml/min). For patients who are not suitable for the treatment mentioned above, they can be treated with conventional chemotherapy, including VAD (vincristine, adriamycin and dexamethasone), MP (mephalan and prednisolone) and high-dose dexamethasone regimen. Adequate hydration (at least 3 litres of fluid intake a day or 2 L·m(-2)·d(-1)) and correcting reversible causes of RI are key points for the supportive care. Renal replacement therapy (more often hemodialysis) should be started in patients with severe AKI and end stage renal disease (ESRD). High flux or high cut-off membrane are recommended because routine hemodialysis could not remove the serum free light chain (sFLC) effectively. Plasmapheresis (PE) is recommended for patients with hyperviscosity syndrome or cast nephropathy presented with AKI, which may help to increase the dialysis-independency.